Genetic instability is now widely recognized as an essential factor in the evolution of cancer (Loeb, 1991; Lengauer et al., 1998). In the vast majority of solid tumors, this instability appears to involve gains and losses of whole chromosomes or large parts thereof, leading to aneuploidy (Lengauer et al., 1997; Duesberg et al., 1999). Recent evidence suggests that this form of chromosomal instability (CIN) is in some cases associated with alterations in a cell cycle checkpoint that monitors the integrity of the spindle apparatus, a structure critical for proper bipolar segregation of duplicated sister chromatids at mitosis (Cahill et al., 1999). A small fraction of CIN cancers are associated with dominant mutations in the human homolog of the yeast spindle checkpoint gene BUB1 (Cahill et al., 1998; Imai et al., 1999; Gemma et al., 2000). Likewise, mutations in the mouse BUB1 gene have been shown to disrupt the mitotic spindle checkpoint (Taylor and McKeon, 1997; Lee et al., 1999). Efforts to study the mitotic spindle checkpoint pathway through genetic approaches have been hampered by the extremely early embryonic lethality of mice homozygously deleted for MAD2 and BUB3, preventing the evaluation of chromosome loss rates in proliferating somatic cells (Dobles et al., 2000; Kalitsis et al., 2000). Recent evidence suggests that disruption of a single MAD2 allele can result in a modest increase in chromosomal instability associated with premature anaphase entry (Michel et al., 2001).
Intensive efforts to dissect the mitotic spindle checkpoint biochemically have elucidated a general mechanism by which BUB1 and other checkpoint proteins arrest mitotic progression in response to spindle damage (reviewed in Amon, 1999; Gardner and Burke, 2000). Specific MAD and BUB proteins are localized to the kinetochores of chromosomes that are unattached to the spindle apparatus (Chen et al., 1996; Li and Benezra, 1996; Taylor et al., 1998; Martinez-Exposito et al., 1999), suggesting that they trigger the checkpoint in cells exposed to microtubule inhibitors or in cells with spontaneously lagging chromosomes. At the biochemical level, the BUB and MAD protein kinase cascade ultimately impinges on a large multiprotein assembly known as the anaphase-promoting complex (APC) that appears to be the master regulator of chromosome segregation and mitotic exit in all eukaryotic cells (for reviews see King et al., 1996; Morgan, 1999; Peters, 1999). Although the mechanisms regulating APC activity are not yet completely understood, it seems clear that at least one outcome of activation of the MAD/BUB pathway is the association of MAD2 with the APC and its accessory factor Cdc20 (Fang et al., 1998). This association inhibits the intrinsic ubiquitinating activity of the APCCdc20 complex, thereby preventing the degradation of securin and later of cyclin B. Activation of the checkpoint thereby delays anaphase and exit from mitosis until all sister chromatids have established bipolar attachments to the spindle apparatus.
The securin proteins are key substrates of the APC pathway and comprise an evolutionarily divergent class of anaphase inhibitors. Members of the securin family include the Pds1 and Cut2 proteins in budding and fission yeast, respectively, the vertebrate pituitary-tumor transforming gene (PTTG or vSecurin) proteins, and the Pimples protein in Drosophila (Cohen-Fix et al., 1996; Funabiki et al., 1996b; Stratmann and Lehner, 1996; Zou et al., 1999). The securins form tight complexes with a well-conserved family of proteins, the ‘sister-separating’ proteases that have been termed separins (reviewed by Nasmyth et al., 2000; Yanagida, 2000). Securin degradation appears to be essential for sister chromatid separation, as expression of non-degradable securins blocks chromosome segregation in both budding and fission yeasts and in animal cells (Cohen-Fix et al., 1996; Funabiki et al., 1996b; Zou et al., 1999; Leismann et al., 2000). Current models propose that securin destruction liberates the active separin protease, allowing it to cleave proteins mediating sister chromatid cohesion, including the cohesin subunit Scc1 (Glotzer, 1999; Uhlmann et al., 1999; Nasmyth et al., 2000; Uhlmann et al., 2000; Waizenegger et al., 2000). This would release tension between paired kinetochores, allowing the separated sister chromatids to migrate poleward along the mitotic spindle.
Paradoxically, there is also evidence that securin plays a positive role in promoting sister separation. In fission yeast, loss of securin is lethal and produces the same effect as loss of separin itself, i.e., a complete block to chromosome segregation and completion of mitosis (Funabiki et al., 1996a). Similarly, Drosophila pimples mutants fail to separate sister chromatids during mitosis 15 (Stratmann and Lehner, 1996). Close examination of pds1 mutants in S. cerevisiae also demonstrates retarded anaphase entry and synthetic lethality with separin mutations (Ciosk et al., 1998), arguing that even in budding yeast, securin and separin may act synergistically rather than purely antagonistically in regulating anaphase.
There is a need in the art for therapeutic agents which are selectively toxic to aneuploid cells relative to euploid cells.